final presentation 11-16-2012. sample preparation nextera truseq ribozero strand specific clontech...

Final Presentation 11-16-2012

Sample Preparation

• Nextera• TruSeq• RiboZero• Strand Specific• Clontech• smRNA• 16s

Nextera

• Quantification issues Nextera libraries consistently have lower qPCR

concentrations compared to BA; usually about half as concentrated

• Illumina Sequences

My Guess?

Nextera Sequences

• CTTAATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCGTGATGGACTGCCGTAACGGCGACCTGCTGTGCATGGCCTCCGCGCCGAGCTTCGACGCCAACCGGTTCGTGCGGGGGCTGTCCGGTCCTGAGTACAAGGCCCTGGCCGAGTACGAGCGCAAGCCGCTGCTCGACAAGTCGATGACCGGCCTGTTTCCGCCCGGCTCGACCTTCAAGCCCACGGTGGGTCTGGCCGCCCTGGCCGCCGGCATCGATCCCGAGGTCCGGGTCAACTGTCCGGGCAGCTGGTACTATGGCGGTCGGGTGTGGCGTTGCTGGGAGAAGGGCGGCCACGGCCTGTCTCTTATACACATCTCCGAGCCCACGAGACTAAGGCGAATCTCGTATGCCGTCTTCTGCTTG

TruSeq

•100ng – 4000ng total RNA input• “yield” ~70%• PolyA based purification• Eukaryotic only• RINs should be greater than 9

RiboZero• 1- 3ug total RNA input, can handle fragmented RNA• rRNA depletion, magnetic beads with capture probes against rRNA• Creates libraries with mRNA and non-coding RNAs• Species specific reagents, prokaryotic and eukaryotic • High rRNA background with input above 3ug; might be worth doing rRNA removal twice

Strand Specific•Requires greater than 1ug total RNA input• dUTP 2nd strand marking protocol• Need to add Actinomycin D during 1st strand synthesis• TruSeq and RiboZero protocols can be used for strand specific preparations

Clontech

• 100pg total RNA input• polyA based purification,

eukaryotic only• Requires RIN greater than 9• Full length cDNA, requires

sonication or treat with Nextera (never tested)

• Reagents have short shelf life ~6 months

smRNA• 1-10ug total RNA, RIN greater than 9• Only 12 barcodes in stock• Requires addition size selection

Pippin Prep

• Software does not consistently call ladder.

• Yeild ~50%• Size selection +- 40bp

Metagenomics/16s Sequencing

• Metagenomics is an emerging field • Genomic analysis is applied to microbial

communities rather than individual microbes• Bypasses the need to isolate and culture individual microbial species• Many microbes are resistant to culturing• Has potential medical uses.

16sTwo PCR steps used to create V4 specific sequence capable

libraries. Dual Barcodes – 1st read 5’ barcode. 2nd read 3’ barcode

V1 V2 V3 V4 V5 V6 V7

Offset PrimersHigh base conservation upstream and downstream of V4 regionDesigned primers to offset sequence to improve base balance

T B A R C O D E T G C C A G C

T B A R C O D E A T G C C A G

T B A R C O D E T C T G C C A

T B A R C O D E C A A T G C C

Upstream – Read 1

Downstream – Read 2

C G A T C T G G A C T A H V G

C G A T C T A G G A C T A H V

C G A T C T C A G G A C T A H

C G A T C T T C T G G A C T A

To Do

• Optimize RiboZero to reduce rRNA contamination

• Test ability to use Nextera instead of covartis fragmentation for clontech samples

• Run 16s offset samples to check base balance